Spectrometric imaging apparatus

ABSTRACT

An inelastic diffusion spectrometric imaging apparatus includes an illuminating and energising system including a confocal first aperture and a second confocal aperture combined with the first. A first deflector assembly scanning for scanning a sample and a second deflector assembly sychronised with the first, are placed respectively downstream and upstream of the second confocal aperture and a spectrometer. The input aperture of the spectrometer merges with the second confocal aperture.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a National Stage entry of International ApplicationNo. PCT/FR00/02278, filed Aug. 8, 2000, the entire specification claimsand drawings of which are incorporated herewith by reference.

This invention relates to a spectrometric imaging apparatus, especiallyof Raman or of low level fluorescence type.

The patent application EP-A1-0.502.752, in the name of the applicantcompany, discloses a confocal scanning spectrometry device comprising:

-   -   an assembly comprising:        -   an illuminating and energizing system including a first            confocal aperture,        -   an optical system,        -   a second confocal aperture combined with the first, and        -   a first and a second synchronized deflector assemblies,            placed respectively downstream and upstream of the second            confocal aperture, and    -   a spectrometer comprising:        -   an input slot,        -   a spectral disperser, and        -   a two-dimensional multichannel detector.

Such a realisation enables to reduce the analysis time of a sampleconsiderably with respect to conventional confocal spectrometry devices.Line scanning of the object can be performed in that the light beamscans the input slot of the spectrometer. The device of that previousapplication also offers an enlargement factor, due to the use of twosynchronous deflectors, which enables to change the dimension of animage scanned in an object space, from the maximum dimension covered bythe lens of the confocal microscopy assembly down to a very smalldimension which is solely limited by the detectable energy threshold.

The content of that previous application EP-A1-0.502.752 must beconsidered as part of this application, for all matters pertaining tothe realisation mentioned above.

The present invention relates to a spectrometry imaging apparatusenabling to extend the usage of the device known by the previousapplication to the whole spectral field useful to Raman spectroscopy andliable to be more readily employable than the previous device.

To that end, the invention concerns a spectrometric imaging apparatuscomprising:

-   -   a confocal microscopy assembly comprising:        -   an illuminating and energizing system including a first            confocal aperture,        -   an optical system,        -   a second confocal aperture combined with the firsts and        -   a first deflector assembly, capable of scanning lines on a            sample and a second deflector assembly, both synchronized,            placed respectively downstream and upstream of the second            confocal aperture, illuminating    -   a spectrometer comprising:        -   a spectral disperser, and        -   a two-dimensional multichannel detector.

According to the invention, the spectrometer comprises an input aperturecombined with the second confocal aperture.

The second deflector assembly is thus arranged downstream of the inputto the spectrometer. This input is a confocal aperture.

The first and the second confocal apertures are preferably composed ofadjustable holes, obtained by means such as transmission or reflectiondiaphragms. They are approximately circular and of small dimensions.

With respect to the known device, described in the applicationEP-A1-0.502.752, the imager of the invention simplifies the assemblywhile reducing the number of optical parts, which is an importantadvantage in particular in the ultraviolet area, and while suppressing aseparate confocal aperture.

Moreover, the spectrometric imaging apparatus according to the inventionhas the advantage of being integrated to a so-called infinite apparatus,involving parallel beams. Thus, the imaging elements of the inventioncan be inserted in the form of blocks in pre-existing devices comprisingan infinite microscope and/or an infinite spectrometer.

Preferably, the spectrometric imaging apparatus of the invention isarranged for Raman spectroscopy or low level fluorescence spectroscopy.

The dispersive spectrometer is preferably stigmatic, i.e. it producesfor each point of the input aperture, a spectral image covering a lineof pixels of a bi-dimensional multichannel detector.

Preferably, the first deflector assembly comprises substantially a-focaloptics and is placed on a parallel beam.

Thus, this first deflector assembly does not induce any signal losses.It enables:

-   -   to deflect a beam as well as    -   to carry pupils over.

The first deflector assembly can be of the refractive or reflective type(i.e. operating respectively in refraction or in reflection).

According to a first preferred embodiment of a-focal optics, the lattercomprise a converging lens, a diverging lens and a first refractivedeflector placed between these lenses.

In a second preferred embodiment of a-focal optics, the latter comprisemirrors and a first reflection deflector capable of receiving a parallelbeam from one of these mirrors and of reflecting this parallel beamtowards another mirror.

The mirrors comprise advantageously spherical mirrors.

In a peculiar embodiment, the first deflector assembly comprises a firstdeflector which, by means of optical elements, is arranged for twodimension scanning on a sample. Notably, in combination with the firstor the second preferred embodiments of the a-focal optics, the firstdeflector is then capable of scanning frames on one sample, in adirection perpendicular to the lines.

In another embodiment, obtained from the first or the second preferredembodiments of the a-focal optics:

-   -   the first deflector assembly comprises translation means for the        lenses or for the mirrors, enabling to scan this line in a        direction perpendicular to that line.

Thus two sub-forms of both first embodiments of the a-focal optics canbe distinguished:

-   -   either the first deflector scans frames perpendicular to these        lines,    -   or the first deflector scans a line and the associated elements        in the a-focal optics scan at right angle to that line; the        deflection obtained by these elements, so-called        frame-deflection, is then much slower than that produced by the        first deflector; called line deflection (the line deflection        frequency is a multiple integer of the frame deflection        frequency).

The arrangement with means for translating the lenses or mirrors enablessimplification of the system while reducing the number of components.

In an embodiment variation, the first deflector is capable of scanning aline on a sample, for example by the rotation of an optical element, andit is itself mounted on a mobile device, enabling to move this line, onthe object, in a direction perpendicular to this line (frame scanning).

It is interesting that the first deflector assembly also generates ascanning along a third dimension, parallel to the beam (in-depthanalysis of the sample) thus enabling the production ofthree-dimensional special confocal images.

The second deflector assembly, placed in the spectrometer, produces adeviation making up a line deflection (perpendicular to the grid linesformed on the multichannel detector), which is synchronous with thatrealised by the first deflector assembly. This second deflector assemblyis preferably reflective. It thus enables to reduce spurious lighteffects. In an embodiment variation, the second deflector assembly isrefractive.

Preferably, the first and second deflector assemblies producesynchronous deflections with variable amplitudes allowing the changingof the enlargement factor.

Advantageously, controlled displacement along the axis of the microscopeenables to generate three-dimensional spectral confocal images.

In a first preferred arrangement of the second deflector assembly, thesaid assembly is placed between the input aperture of the spectrometerand the multichannel detector.

Thus, in an embodiment of this first arrangement, the spectrometercomprises a first lens or collimator and the second deflector assemblyis placed between the input aperture and this first lens. Thisembodiment is easy to implement.

In another variation of this embodiment, the spectrometer comprises afirst spherical mirror upstream of the spectral disperser and the seconddeflector assembly is placed between the input aperture and thisspherical mirror.

In an arrangement of the second deflector assembly, the said assembly isplaced between the spectral disperser and the multichannel detector.

In a second arrangement of the second deflector assembly, the saidassembly comprises at least a portion of the spectral disperser itselfReflection can then be obtained by oscillation of the spectral disperseror of one of its optical elements or by a device combined with the saiddisperser.

According to a third arrangement of the second deflector assembly, thesaid assembly consists of the multichannel detector. The seconddeflector assembly uses then the charge transfer on a charge-coupleddevice of the multichannel detector. It is provided advantageously thatthe detector should have a sequential control and that the firstdeflector assembly has a ‘stepping’ control, in order to obtainsynchronisation of both deflector assemblies easily.

According to an advantageous embodiment, the imager comprises an opticalfibre placed between the first and the second synchronous deflectorassemblies, coupled to the input aperture of the spectrometer andintended for remote transport of the information enabling to build aconfocal space image.

Thus, the second confocal aperture can be projected onto the opticalfibre that carries the information. The scanning phase should then betransported or controlled in order to ensure synchronisation of thedeflectors.

The invention will be better understood and illustrated using nonlimitative examples with reference to the appended drawings on which:

FIG. 1 represents a general diagram of a first embodiment of aspectrometric imaging apparatus according to the invention and its use;

FIG. 2 illustrates a first embodiment of the first deflector assembly ofthe imager of FIG. 1;

FIG. 3 illustrates a second reflective embodiment of the first deflectorassembly of the imager of FIG. 1;

FIG. 4 is a simplified perspective representation of the deflector ofthe first deflector assembly of FIG. 3;

FIG. 5 is a front representation of the deflector of the first deflectorassembly of FIG. 3;

FIG. 6 is a lateral view of the deflector of the first deflectorassembly of FIG. 3;

FIG. 7 shows a third embodiment of the first deflector assembly of theapparatus of FIG. 1 and

FIG. 8 illustrates a second embodiment of a spectrometric imagingapparatus according to the invention.

A spectrometric imaging apparatus according to the invention comprises amicroscopic illuminating system 1 and a spectrometer 2. The microscopicilluminating system 1 comprises an energising source such as a laser 10associated with a first confocal aperture, such as a size- andposition-adjustable hole. The system 1 also contains a microscope lens11, towards which is directed a laser beam emitted by the laser 10 afterspace filtering by the aperture 15, reflection on a partially reflectingseparator 12 and reflection on a plane mirror 13. The system 1 alsocomprises a first deflector assembly 31, enabling to scan a sample 3along one, two or three dimensions.

The system 1 also has a second optical system guiding a beam emitted bythe sample 3, collected by the microscope lens 11 and transmitted by theseparator 12, to the spectrometer 2. This second optical systemcomprises, for example, converging optics 14.

The spectrometer 2 is a multichannel spectrometer fitted with abidimensional detector 23. Most often, the bidimensional detector isassociated with processing means that collect the signals produced bythe detector in real time and that may give a representation thereofeither in real time or in delayed time. In the text below, we shalldesignate generally by the term detector the photoelectric converteritself and the associated processing means. The spectrometer comprisesan input aperture 25, which forms a second confocal aperture combinedwith the first confocal aperture 15. A confocal aperture refers to asmall hole, substantially circular, formed in a cache. The secondconfocal aperture 25 plays therefore in the spectrometer the usual partof a slot in the grid spectrometers.

The spectrometer also comprises a first optical system 21 or collimatorand a spectral dispenser 22, in the form of a diffraction grid. Suchdiffraction grid is formed of lines parallel to each other. A parallellight beam incident on the grid is dispersed on the dispersion planethat is perpendicular to the lines of the grid. The spectrometer isfitted with a second deflector assembly 32, arranged in the examplerepresented between the input aperture 25 and the optics 21.

In operation, a laser beam emitted by the laser 10 and deviated by thedeflector assembly 31 is sent towards the sample 3. This beam scans aline 40 of the sample 3. The light Raman flux diffused in succession byeach point of this line 40 of the sample is collected by the lens of themicroscope 11 and focused, after passing through the first deflectorassembly 31, into an input point of the spectral disperser 22.

The second deflector assembly 32, synchronised with the first produces ascanning parallel to the lines of the grid and addresses the lightspectrum 43 generated from each point of the line 40 scanned on a lineof the detector 23.

We can thus obtain on the detector 23 vertically, for example, thespatial distribution of the line 40 (line 41) and horizontally, thespectral data 43 (line 42).

The second deflector assembly 32 is in practice adjusted so that itsdeflection on the detector 23 corresponds to the vertical dimension ofthe said detector and that the first deflector assembly 31, synchronouswith the deflector assembly 32, is adjusted between zero and a maximumvalue limited by the field of the lens (whereas the zero valuecorresponds to the analysis of a point).

The first deflector assembly 31 comprises a-focal optics. In a firstembodiment of this a-focal optics (FIG. 2), the first deflector assemblyreferred as 31A comprises a refractive deflector 50 placed between aconverging lens 51 and a diverging lens 52. The lenses 51 and 52 haverespectively focal distances close to one another. The first deflectorassembly 31A is such that an incident parallel beam 55 comes out in theform of a deviated quasi-parallel beam 56. In this embodiment, thelenses 51 and 52 are fixed and the deflector 50 produces the deflectionsrequested.

In a second embodiment of the first deflector assembly 31, referred 31B(FIG. 3), the said deflector comprises a reflective deflector 60 and twospherical mirrors 61 and 62, arranged for example respectively upstreamand downstream of the deflector 60. The deflector 31B may also comprisetwo plane mirrors 63 and 64 arranged respectively upstream anddownstream of the spherical mirrors 61 and 62. The whole forms a-focaloptics in association with the deflector 60 and thus provides a parallelbeam 56 from a parallel beam 55 at input.

The mirrors 61–64 are fixed, while the deflector 60 performs therequested scanning cycle(s).

Preferably, the deflector 60, which comprises for instance, a planemirror 70, is mobile in rotation around a first axis 71, enabling framescanning (i.e. parallel to the lines of the refraction grid on themultichannel deflector 23) and around a second axis 72, enabling linescanning (i.e. scanning perpendicular to the lines of the diffractiongrid on the multichannel detector 23) (see FIG. 4). In an embodiment ofthis deflector 60 (FIGS. 5 and 6), the said deflector comprises a motor73 actuating into rotation the mirror 70 around the axis 71 via a shaft74, and a second motor 76 actuating the mirror 70 into rotation aroundthe second axis 72, for example using balls 77 and 78 arranged laterallywith respect to the mirror 70, and an elastic recall device acting as apart 75 arranged above the mirror 70. This motor 76 is then preferably astepping motor.

When the first deflector scans the object in two dimensions, the seconddeflector is always synchronised on the scanning of this object in adirection, for example the line scanning. The detector then generates insuccession the spectral information from the different lines. Thesepieces of information are captured by an associated processing unit thatis capable of presenting the information of the spectrum diffused by thepoints of both dimensions of the sample in any requested format. It canbe understood that any means enabling to scan the object in threedimensions can be processed in a similar fashion. The scanning of thetransversal plane has been described as a line and frame process, whichis preferable, but any scanning of the complete plane enables to realisean image.

In a third embodiment of the first deflector assembly 31, referred 31C(FIG. 7), the said assembly comprises a refractive deflector 80 placedbetween two lenses 81 and 82, respectively converging and diverging. Thelenses 81 and 82 and the deflector 80 being aligned along an axis 84,the deflector 80 is mobile in rotation around a rotational axis 85perpendicular to the axis of alignment 84, in order to allow linescanning. Moreover, the first deflector assembly 31C comprisestranslation means 83 for the lenses 81 and 82 along a direction 86parallel to the axis of rotation 85, for frame scanning, which is muchslower than the line scanning (the line deflection frequency is amultiple integer of the frame deflection frequency). This firstdeflector assembly 31C thus forms a compact a-focal system comprising areduced number of components, and providing a parallel beam 56 from anincident parallel beam 55.

In a fourth embodiment of the first deflector assembly (notrepresented), the second embodiment 31B of the first deflector assemblyis adapted in a similar fashion, while making the spherical mirrors 61and 62 mobile, which enables to limit the deflection movements of thedeflector 60 to line scanning only.

In a second embodiment of the spectrometric imaging apparatus (FIG. 8),identical or similar elements being designated by the same references,this apparatus comprises a confocal microscopy system 101 and aspectrometer 102. The spectrometer 102 comprises, besides the inputaperture 25, the collimator 21, the spectral disperser 22 and themultichannel detector 23, an optical fibre 26 coupling the collimator 21to a second lens 27 at the output of the optical fibre 26, a diaphragm28 and the second deflector assembly 32, arranged between the spectraldisperser 22 and the detector 23.

Thus, the optical fibre, placed between the first and second deflectorassemblies ensures remote transport of the information enabling to builda confocal spectral image.

The second deflector assembly 32, synchronous with the first deflectorassembly 31, is placed downstream of the input aperture 25. In certainembodiment variations, it is situated between the input aperture 25 andthe disperser 22, on the disperser 22 or combined with the said, betweenthe disperser 22 and the detector 23 or on the detector 23 or combinedwith the said.

Particularly advantageous embodiments are given by the followingcombinations. In the cases when the second deflector assembly 32 isplaced between the input aperture 25 and the disperser 22 or on thedisperser 22 or still between the disperser 22 and the detector 23 or onthe detector 23, we choose generally the first embodiment of the imager(FIG. 1), which enables to work on a beam smaller than the secondembodiment (FIG. 8) and we use preferably reflection operatingdeflectors (FIGS. 3 to 6).

In case when the second deflector assembly 32 is combined with thedisperser 22, we use preferably mechanic or piezoelectric deflectors.Indeed, the angular deflection is then small.

1. A spectrometric imaging apparatus, comprising: a microscopicilluminating assembly, comprising: an energizing source; a firstconfocal aperture; an optical system; and a first deflector assemblyconfigured to scan lines on a sample; and a spectrometer, comprising: asecond confocal aperture which forms an input aperture of thespectrometer, wherein the second confocal aperture is coupled to thefirst confocal aperture; a second deflector assembly, wherein the firstdeflector assembly and the second deflector assembly are synchronized,the first deflector assembly is positioned upstream of the secondconfocal aperture, and the second deflector assembly is positioneddownstream of the second confocal aperture; a spectral disperser; and atwo-dimensional multichannel detector, wherein the microscopicilluminating assembly is configured to illuminate the spectrometer. 2.The spectrometric imaging apparatus of claim 1, wherein the firstdeflector assembly is configured to scan frames on the sample in adirection which is perpendicular to the said lines.
 3. The spectrometricimaging apparatus of claim 1, wherein the first and second deflectorassemblies produce synchronous deflections with variable amplitudesallowing a changing in an enlargement factor; the enlargement factorbeing created by the use of two synchronous deflectors, wherein theenlargement factor allows for a change of at least one dimension of animage scanned in an object space.
 4. The spectrometric imaging apparatusof claim 1, wherein the second deflector assembly is positioned betweenthe second confocal aperture and the multichannel detector.
 5. Thespectrometric imaging apparatus of claim 1, further comprising anoptical fiber positioned between the first and the second deflectorassemblies and coupled to the second confocal aperture and configured toallow for remote transport of information that allows for a generationof a confocal spatial image.
 6. The spectrometric imaging apparatus ofclaim 1, wherein the first deflector assembly comprises substantiallya-focal optics and is positioned on a parallel beam.
 7. Thespectrometric imaging apparatus of claim 6, wherein the a-focal opticscomprises a converging lens, a diverging lens, and a first refractiondeflector positioned between the converging lens and the diverging lens.8. The spectrometric imaging apparatus of claim 6, wherein the a-focaloptics comprises a first mirror; a second mirror; and a reflectiondeflector configured to receive the parallel beam from the first mirrorand to reflect the parallel beam toward the second mirror.
 9. Thespectrometric imaging apparatus of claim 7, wherein the first deflectorassembly comprises translation means for the converging lens and thediverging lens, wherein the translation means allows the first deflectorassembly to scan the lines in a direction which is perpendicular to thelines.
 10. The spectrometric imaging apparatus of claim 8, wherein thefirst deflector assembly comprises translation means for the firstmirror and the second mirror, wherein the translation means allows thefirst deflector assembly to scan the lines in a direction which isperpendicular to the lines.